SCIENCE ADVANCES | RESEARCH ARTICLE APPLIED SCIENCES AND ENGINEERING Copyright © 2021 The Authors, some Discovery of a natural cyan blue: A unique food-sourced rights reserved; exclusive licensee anthocyanin could replace synthetic brilliant blue American Association for the Advancement of Science. No claim to Pamela R. Denish1,2†, Julie-Anne Fenger3†, Randall Powers4†, Gregory T. Sigurdson5, original U.S. Government Luca Grisanti6,7, Kathryn G. Guggenheim8, Sara Laporte6, Julia Li4, Tadao Kondo9, Works. Distributed Alessandra Magistrato10, Mícheál P. Moloney3, Mary Riley11, Mariami Rusishvili6,12, under a Creative Neda Ahmadiani5,13, Stefano Baroni6,9, Olivier Dangles3, Monica Giusti5, Thomas M. Collins4, Commons Attribution John Didzbalis14, Kumi Yoshida9*, Justin B. Siegel1,2,8,15*, Rebecca J. Robbins16* License 4.0 (CC BY). The color of food is critical to the food and beverage industries, as it influences many properties beyond eye-pleasing visuals including flavor, safety, and nutritional value. Blue is one of the rarest colors in nature’s food palette— especially a cyan blue—giving scientists few sources for natural blue food colorants. Finding a natural cyan blue dye equivalent to FD&C Blue No. 1 remains an industry-wide challenge and the subject of several research pro- grams worldwide. Computational simulations and large-array spectroscopic techniques were used to determine the 3D chemical structure, color expression, and stability of this previously uncharacterized cyan blue anthocyanin-­ based colorant. Synthetic biology and computational protein design tools were leveraged to develop an enzy- matic transformation of red cabbage anthocyanins into the desired anthocyanin. More broadly, this research demonstrates the power of a multidisciplinary strategy to solve a long-standing challenge in the food industry. INTRODUCTION (8). Although green is abundant in nature, the chlorophyll chromo- Despite a long history of exploration, blue remains one of the most phore is not stable or water soluble (section S2.1) and, therefore, has challenging colorants to obtain from any source and even more so limited application (12). Achieving a cyan blue from natural sources from natural, edible sources (1–7). Blue is critical as it is necessary that could be used as replacement for FD&C Blue No. 1 enabling a broader to produce other colors across the palette. Furthermore, the subtle- color palette has been a long-standing challenge to the food industry. ties of color differences in different blues are of great importance in Anthocyanins display a large versatility in color expression be- the area of blending colors (8). The two main artificial blue food cause of a complex chemical equilibrium of colored and colorless forms colorants are brilliant blue (FD&C Blue No. 1) and indigotine (FD&C (fig. S3.1 and discussion S3.1) (13–17). These naturally occurring chro- Blue No. 2), providing cyan (max = 630 nm) and indigo (max = 608 nm) mophores are generally intensely red-colored under acidic conditions hues, respectively. Naturally occurring food-based blues are limited based on the flavylium cation (Fig. 1B) and change toward violet and can be sourced from anthocyanins and a limited set of other blue and blue as the pH increases. Concurrently, the colored forms are colorants including phycocyanins (from Spirulina spp.) (max = 615 to susceptible to fading by a combination of water addition (reversible) 620 nm) (9) and iridoid derivatives from huito (or gardenia) (max = 590 and autoxidation (irreversible), limiting their stability over time (18). to 610 nm) (10, 11). However, to date, all known natural colorants A challenge with anthocyanins is the plethora of different molecules have either a max less than 630 nm, a large violet color contribution found in a single crop source, such as red cabbage (Fig. 1B) or pur- (absorbance in the range of 500 to 600 nm), or both (Fig. 1A, method ple sweet potato (19, 20). Both well-established commodity crops S1.1, and figs. S1.1 and S1.2). Violet contributions affect the final generate blue colors at pH 7 to 9. However, it is a composite color color in blended colorants, e.g., blending natural blues with yellow because of the numerous individual anthocyanin molecules present generally results in a muddy green (fig. S2.1 and discussion S2.1) and the various equilibrium forms at a given pH for each individual anthocyanin molecule (10, 19, 20). While the combined red cabbage 1 Biophysics Graduate Group, University of California, Davis, Davis, CA, USA. 2Genome anthocyanins (RCAs) will produce a vibrant and attractive blue color Center, University of California, Davis, Davis, CA 95616, USA. 3Avignon University, in pH-neutral solution, there is still a relatively large violet color con- INRAE, Avignon, France. 4Mars Wrigley, Hackettstown, NJ 07840, USA. 5Department tribution, limiting their utility as a natural FD&C Blue No. 1 replace- of Food Science and Technology, The Ohio State University, Columbus, OH 43210, ment (Fig. 1A). USA. 6Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy. 7Division of Theoretical Physics, Institut Ruđer Bošković, Zagreb, Croatia. 8Chemistry Depart- Previous efforts to identify the chemical structures and spectral ment, University of California, Davis, Davis, CA 95616, USA. 9Graduate School of properties of RCAs uncovered an interesting structural homology where Informatics, Nagoya University, Chikusa, Nagoya, Japan. 10Consiglio Nazionale delle the common building blocks are a cyanidin chromophore, a sophorose Ricerche, Istituto Officina dei Materiali, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy. 11Microbiology Graduate Group, University of California, Davis, moiety bound at position 3, and a glucose bound at position 5 (Fig. 1B) Davis, CA 95616, USA. 12Pritzker School of Molecular Engineering, The University of (21–25). The key differentiation factors are the presence of hydroxy- Chicago, Chicago, IL, USA. 13Centre d’Innovació, Recerca I Transferència en Tecnologia cinnamic acids and their substitution patterns on sugar 1 (Glc-1) or dels Aliments, CERTA-UAB Tecnio Grup, XIA-UAB, Animal and Food Science Depart- ment, Universidad Autònoma de Barcelona, Bellaterra, Spain. 14Mars Advanced sugar 2 (Glc-2) of the sophorose (Fig. 1B, table inset). Remarkably, Research Institute, Mars, Incorporated, Hackettstown, NJ 07840, USA. 15Department one of the minor mono-acylated anthocyanins, Peak 2 (P2) was found of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, to have a particularly high max of 640 nm at pH 7 (20). While only CA 95616, USA. 16Mars Wrigley Global Innovation Center, Chicago, IL 60642, USA. a minor component within the RCA mixture, this unique compound *Corresponding author. Email: yoshidak@i.nagoya-u.ac.jp (K.Y.); jbsiegel@ucdavis. edu (J.B.S.); rebecca.robbins@effem.com (R.J.R.) warranted further investigation to understand the structural factors †These authors contributed equally to this work. conferring its desirable spectral properties. Denish et al., Sci. Adv. 2021; 7 : eabe7871 7 April 2021 1 of 8 SCIENCE ADVANCES | RESEARCH ARTICLE RESULTS AND DISCUSSION Structural analysis of P2 Previous efforts relied on mass and retention time or established standards to make predictions of the P2 structure (20). Before con- ducting an in-depth investigation of the structural factors, which impart the unique spectral properties to P2, it was prudent to obtain a complete structural assignment of the compound. The structural assignment for P2 was determined by high-resolution electrospray ionization–time-of-flight mass spectrometry (ESI-TOF-MS) and a combination of 1H and 13C nuclear magnetic resonance (NMR), one-dimensional total correlation spectroscopy (1D TOCSY), cor- relation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear single-quantum correlation spectroscopy (HSQC), and heteronuclear multiple bond correlation (HMBC) experiments and proven to be 3-O-(2-O-(2-O-(E)-sinapoyl--d-­ glucopyranosyl)--d-glucopyranosyl)-5-O--d-glucopyranosyl­ cyanidin (discussion S5.1, table S5.1, and figs. S5.1 to S5.9) (26, 27). The results were consistent with the previously predicted structure, supporting that P2 is a regio-isomer of P5 where only the sinapoyl residue resides on a different sugar (Fig. 1B). It is well established that cyanidin-based chromophores, such as P2, require a combination of neutral pH and complexation to a metal ion, such as an Al3+, at the ortho-dihydroxyphenyl moiety (Fig. 1B, positions 3′ and 4′) of the cyanidin chromophore to produce a blue color (15, 17, 27, 28). To evaluate the effect of metal ion complex- ation with specific RCA components, P2, P5, and P8 were incubated with Al3+. The only difference between P2, P5, and P8 is the number and placement of sinapoyl moieties on the sophorose, enabling a direct comparison of the primary structural feature differentiating RCAs and their effect on color formation. The discrimination between the spectral properties of P2, P5, and P8 in the presence of Al3+ is highly unexpected (Fig. 2A). With only one-third equivalent of Al3+, the P2 solution gave the desired blue colored complex with a large bathochromic shift of >40 nm (max at 640 nm; Figs. 1A and 2A, figs. S6.1 to S6.4, and discussion S6.2). In contrast, P8 and P5 had only modest shifts of <20 nm. This provides direct evidence that the sinapoyl moiety and its position (on Glc-2 in P2), despite its intrinsic inability to bind metal ions, play a critical role in the structure of the metal complex, leading to a very strong bathochromic shift. To elucidate this uniquely colored self-assembled structure of the three P2 molecules coordinated to aluminum metal (29), the circu- lar dichroism (CD) spectrum of P2 and Al3+ was recorded in buff- ered solutions. With the addition of Al3+, P2 showed an exciton-type positive Cotton effect around max, while no such Cotton effect was observed without Al3+. These results indicate that Al3+ is critical to Fig. 1. Color expression of four blue pigments and the structure of RCAs. (A) The the coordination and building of the tertiary structure that exhibits UV-visible spectra depicted highlights max and its respective location on the elec- a chiral arrangement of three P2 monomers around the Al3+ ion (Fig. 3, A tromagnetic (EM) absorbance spectra. max for FD&C Blue No. 1 is 630 nm, spirulina and B, and section S8). By contrast, P5 displayed very minor Cotton is 617 nm, RCAs at pH 8 are 608 nm, and the Al3+(P2−)3 complex at pH 7 is 640 nm. The effects (section S7.1, figs. S7.1 to S7.3, and table S7.1). Furthermore, violet contribution (VC) of each blue, defined as the absorption in the 500- to 600-nm negative-mode high-resolution ESI-TOF-MS analysis of the P2/Al range (depicted in gray), is shown. Values and calculations for VC, i.e., area under solution at pH 7 provided evidence for molecular ion peaks at mass/ the curve (AUC), are provided in sections S1 and S2. (B) The red flavylium cation of charge ratio (m/z) = 1478.8629 and at m/z = 985.5760, which can the main anthocyanins in red cabbage is depicted as P1 to P8 on the HPLC-diode-­ be respectively attributed to [3×P2+Al−2H]2− and [3×P2+Al–3H]3−, array-detection (DAD) trace (520-nm detection) (method S11.2). RCAs display a struc- tural homology (red and blue arrows) where the common building blocks are a cyanidin indicating the formation of a Al3+-P2 complex of 1:3 stoichiometry chromophore (highlighted in red), sugars, and small acyl groups (highlighted in blue). (Fig. 3A, section S7.2, and figs. S7.4 to S7.6). P5 did not give such The sophorose moiety is bound at position 3, and a glucose is bound at position 5. multivalent molecular ion peaks by addition of Al3+ (fig. S7.5 to The key differentiation is acyl groups (p-coumaroyl, feruloyl, and sinapoyl) and their S7.7); however, P8 showed the similar divalent and trivalent ion substitution patterns on sugar 1 (Glc-1) or sugar 2 (Glc-2) of the sophorose. peaks of [3×P8+Al–2H]2− and [3×P8+Al–3H]3− (figs. S7.8 to S7.10). Denish et al., Sci. Adv. 2021; 7 : eabe7871 7 April 2021 2 of 8 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 2. Absorption spectrum and stability sinapoyl containing RCAs. (A) Cuvettes with the corresponding visible spectrum, 400 to 700 nm, for P2, P5, and P8 at pH 7 with and without Al3+. The different equivalences of Al are color-coded: black = 0 eq, red = 1/3 eq, and blue = 1 eq. The max values for 0 eq are 598 nm for P2, 596 nm for P5, and 598 nm for P8. Adding 1/3 eq Al3+ generates a max value of 640 nm for P2, 602 nm for P5, and 617 nm for P8. Adding 1 eq of Al3+ does not appear to affect max for P2, P5, or P8. (B) Stability (absorbance at max of aqueous solutions monitored over time) of anthocyanins P2, P5, and P8 at pH 7 over time with (solid line) and without Al3+ (dashed line). The addition of Al3+ enhances the stability of both P2 and P8; however, it has a minor effect on the stability of P5. P8 has an inherent additional stabili- ty because of diacylation but is still surpassed by the stability of P2 + Al3+. Once the Al3+(P2−)3 complex is formed, it demonstrates a marked increase in stability that is thought to stem from the intermolecular interlocking of the P2 moieties (figs. S8.2 to S8.5). Fig. 3. Proposed trimeric structure. (A) A proposed trimeric structure is shown in (B), where three P2 molecules form a propeller-like structure around the aluminum ion. NMR, MS, and Cotton effects of Al3+(P2−)3 were confirmed (details provided in sections S7, S8, and S10). Computational modeling indicates that the large bathochromic shift observed stems mainly from the enhanced distortion of angle , highlighted in blue on the molecular structure. Inset shows the calculated effect of max as a function of  angle distortion. The color of each circle is the average expected color, the size of the marker corresponds to average oscillator strengths, and error bars are comput- ed as SD of max. The data for the figure correspond to cyanidin-3-glucoside in fully solvated water. Molecular structures were pruned by replacing the sugar with a methyl group and absorption spectra calculated with time-dependent density functional theory [6-311G(d,p) at B3LYP] (DFT-B3LYP) and fully detailed in section S10. (B) 3D representation and coordination of the anthocyanin P2 around the aluminum. 1 H NMR measurement of the trimeric complex of Al3+(P2−)3 in by computational modeling. After extensive classical and first-principles D2O gave a very broad spectrum. However, numbers of signals in molecular dynamics (MD) (9) simulations of P2, plausible geome- the lower field were relatively simple, indicating that the arrange- tries were constructed for threefold symmetric propeller-like structures ment of the three P2 ligands within the Al complex might be highly of three P2 molecules around a single aluminum ion and minimized symmetrical. Several signals were assignable, but analysis of NOE with first-principles calculations (discussion S8.1, method S8.1, fig. correlations was difficult (discussion S7.3, figs. S7.11 to S7.15, and S8.1, and table S8.1). In all proposed configurations, the structure of table S7.2). Therefore, a structural analysis of Al3+(P2−)3 was undertaken the P2 cyanidin chromophore is distorted by a torsional displacement Denish et al., Sci. Adv. 2021; 7 : eabe7871 7 April 2021 3 of 8 SCIENCE ADVANCES | RESEARCH ARTICLE of the bond between the B and C rings (Fig. 3A,  angle, and fig. any acyl group bound to Glc-1 of the sophorose moiety while leav- S8.2, inset). A very interesting and remarkable result from the MD ing the sinapoyl group on Glc-2 intact, thus converting P6, P7, and simulations, coupled with first-principles evaluation of excited states, P8 into P2 (Fig. 4, A and B). Initial database mining efforts comprised is that torsional displacement of this bond causes the greatest change screening a broad range of genes encoding hydrolytic enzymes, in- in the simulated color expression (30, 31). The - electronic inter- cluding lactonases, esterases, lactamases, and hydrolases, curated action between acyl and cyanidin motifs is not the main cause of the from the BRENDA database across 26 enzyme classifications (ECs) large color shift, as typically suggested. In this case, the HOMO and (Fig. 4C and tables S11.1 and S11.2) (32). Over three rounds of ge- LUMO energies are more impacted by distortion of the cyanidin nomic mining, a total of 46 genes were tested, of which 17 were ac- nucleus and the interactions among the ones coordinated to the tive on P6 to P8, converting them to P2 (method S11.2). The median same metal (excitonic effects) than by acyl-cyanidin interactions sequence identity of the total set of tested enzymes is 7.0%, and the (discussions S8.1 and S8.2). However, the - interaction between median sequence identity of active enzymes is 13.0% (Fig. 4C), high- the hydrophobic aromatic rings of the acyl and cyanin groups has a lighting the plasticity of functionality within the esterase family through critical influence on the conformations: While intramolecular - the diversity of sequences capable of catalyzing this reaction. stacking dominates in isolated P2 and P5 (discussion S8.3), the pro- The most active of these enzymes was 1AUR, which in its native posed Al3+(P2−)3 complex manifests intermolecular - stacking form hydrolyzes longer fatty acid chains but has demonstrated broad between adjacent P2 moieties (interlocking between P2 units shown specificity toward esters (fig. S11.1) (33). We subsequently rede- in figs. S8.2 to S8.4), which is consistent with the strong Cotton ef- signed the pocket of the 1AUR protein (24) using a combination of fect observed by CD and the enhanced stability of the complex dis- Rosetta Design and FoldIt to introduce new favorable interactions cussed below. with P8 (the largest and most complex of the RCAs) (34, 35). Across Among all RCAs, P2 uniquely displays two remarkable long- multiple rounds of design, the mutant with the best conversion of range NOE correlations between the cyanidin and sinapoyl residue P6, P7, and P8 was M73H, which was able to drive the transforma- in a solvent that does not favor -stacking interactions (MeOH), tion of RCAs 3 to 8 to P1 and P2 to completion in a time frame and indicating a folding of the molecule (figs. S5.3 and S5.4) (21). The enzyme concentration viable for the gram-scale production of P2 disaccharide in the -d-Glc(2-sinapoyl)-1,2--d-Glc-3-cyanidin se- needed for food-product prototyping (Fig. 4B, table S11.3, and figs. quence within monoacylated P2 likely provides optimal intramolecular S11.2 and S11.3). On the basis of the modeling and design efforts, spacing for enhanced conformational stability. Protection against we hypothesize that the two additional hydrogen bonds that are de- hydration (water addition at position C2) was modest with mono- veloped by the histidine residue substituted at position 73, which acylated P5 (pK′h = 2.7, versus 2.1 for nonacylated P1) but more are both exposed at the active cleft, strengthen the enzyme-substrate pronounced with diacylated P8 (pK′h = 3.7) (15, 21, 28). P2 deviates interaction (Fig. 4D). from this trend and is exceptionally resistant to water addition (pK′h = 4.4) despite its monoacylated status (section S9.1, fig. S9.1, and table S9.1) Color expression and application of P2 (14, 15). This resistance to hydration is reflected by the superior long-­ Following chromatographic purification of the enzymatic transfor- term stability of P2 compared to P5 and P8 and Al3+(P2−)3 com- mation and complex formation with Al3+ (methods S11.3 to S11.4 pared to the P5 and P8 mixtures (Fig. 2B and fig. S6.3). In addition, and section S12), gram quantities of the Al3+(P2−)3 complex were although MD simulations of P2 and P5 show that, in both cases, obtained, enabling detailed investigations of the color expression and folded conformations are favored, P2 displays a higher percentage stability of the novel colorant. In any food product application, col- of closed conformations than P5 (method S10.1, fig. S10.1, and dis- or stability is critical. While many naturally sourced colorants, in- cussion S10.1). It is hypothesized that the “tightly closed” P2 structure cluding anthocyanins, have limited stability over time, the Al3+(P2−)3 enables a closer approach to Al3+ (fig. S10.1c) and a trimeric ar- complex at pH 7 showed remarkable stability in sugar syrup over 55 days rangement around the metal ion. This arrangement causes an in- with only a 14% loss of color (Fig. 5A) (19). Its performance as a colo- crease in the torsional distortion (angle ) of the B-ring to C-ring rant was also demonstrated in application to create blue and green bond, which enhances the bathochromic shift of the visible absorp- colors (hue angle match to a Christmas green shade; fig. S2.1) in tion band (Fig. 3A, inset). Although P8 is closely related to P2, its several food and confectionery products (Fig. 5, B and C, figs. S13.1 additional sinapoyl residue might provide steric hindrance limiting to S13.4, and table S13.1 to S13.4). The stability of this novel colo- the simultaneous interaction of three P8 molecules with Al3+. As for rant in these product applications is excellent as well, with no notable P5, its more open structure (cyanidin-sinapoyl interactions weaker color decay over a 30-day period when stored at ambient conditions than in P2) might prevent the arrangement of anthocyanin mole- (fig. S13.5 and table S13.5). Storage under acidic conditions further cules around Al3+, precluding formation of the Al3+(P5−)3 complex. enhances the stability of the colorant by reducing fraction of hydro- lyzed species, although neutral pH must be able to be restored for Enzymatic enrichment of P2 integration into product (figs. S3.1 and S9.1) (19). While these ini- While the Al3+(P2−)3 complex has the unique chemical conforma- tial studies provide a clear starting point for the development of a tion that provides the spectral properties desired for replacing FD&C natural FD&C Blue No. 1, future efforts will be critical to evaluate Blue No. 1, P2 represents <5% of the total anthocyanin content nat- both stability and color in a wide variety of applications to define urally occurring in red cabbage. Hence, an ambitious challenge was usage limitations (discussion S12.1) and appropriate colorant and obtaining sufficient quantities of P2 at high purity for the present food safety precautions (36–38). research program and for potential use as a food coloring agent. To In summary, combining modern techniques from analytical chem- that end, the homology of structures in red cabbage was leveraged istry, food science, biochemistry, synthetic biology, color science, and and a hydrolytic enzyme capable of a highly selective catalytic deacyla- computational chemistry, we found, characterized, and defined a tion was found and further developed. The enzyme selectively removes path to production for a naturally sourced cyan blue colorant whose Denish et al., Sci. Adv. 2021; 7 : eabe7871 7 April 2021 4 of 8 SCIENCE ADVANCES | RESEARCH ARTICLE Fig. 4. Enzyme discovery and design for transforming RCAs into P2. (A) Structural similarities between RCAs facilitate enzymatic enhancements of P1 and P2. Selective hydrolysis cleaving the acyl group from Glc-1 (red highlight) transforms P3, P4, and P5 into P1, and P6, P7, and P8 into P2. Selectivity toward the acyl group on Glc-1, but not the acyl group on Glc-2 (blue highlight), is critical for P2 production. (B) HPLC chromatograms of RCAs treated with different enzymes: RCAS without enzyme (red, top), RCAs after incubation with the native 1AUR WT protein (blue, middle), and RCAs after incubation with mutant 1AUR M73H (black, bottom). See method S11.2 for reaction conditions. (C) Phylogenetic tree of enzymes tested for activity on RCAs. Black triangles indicate that enzyme had desired hydrolytic activity. Additional metadata are provided in the tree and corresponding legend. See method S11.1 for methodology. (D) (Top) Cartoon depiction of 1AUR WT with P6 in the active site (yellow with gray highlight), with the protein backbone shown in blue. The predicted molecular interactions of the native amino acid (bottom left) and designed mutation (bottom right) with P6 are illustrated. Dashed lines from the introduced histidine depict the predicted new hydrogen bonds between the enzyme and anthocyanin, which we hypothesize contributes to the enhanced activity. Fig. 5. Product prototyping. (A) Visible absorption spectra of Al3+(P2−)3 at pH 7 (solid lines) and RCAs at pH 8 (dashed lines) in saturated sugar syrup monitored over time. Over the course of 10 days, the absorbance at max of RCAs drops by 57%, indicating loss of cyanidin chromophore, whereas the Al3+(P2−)3 complex shows remarkable stability with only 14% loss of absorbance at max over the course of 55 days. Experimental details are in section S13.3. (B) Sugar-coated lentils (top) using the four blue colorants were obtained under typical panning conditions. The colorants were FD&C Blue No. 1, spirulina, RCAs at pH 8, and Al3+(P2−)3 at pH 7. The colorimetry data are plotted in a*b* space and L* values in the sidebar. The Al3+(P2−)3 complex at pH 7 provides a very similar hue angle to FD&C Blue No. 1 (sections S1 and S13). (C) Sugar-­ coated lentils (top) where the colorant blends used were synthetic green, spirulina, red cabbage at pH 8, and Al3+(P2−)3 at pH 7, with the latter three mixed with safflower (Saf) as the yellow component. Al3+(P2−)3 at pH 7 with safflower provides a very similar hue angle to synthetic green (fig. S13.4 and table S13.4). color properties are nearly identical to those of the industry standard MATERIALS AND METHODS brilliant blue FCF (FD&C Blue No. 1). This colorant is also capable Detailed methods for the experiments conducted in this study can of producing superior green colors compared to many existing nat- be found in the Supplementary Materials. ural blue colorants. This discovery requiring a global collaboration provides a solution to a long-standing food color need, potentially Violet contribution calculation fulfilling the growing consumer demands for utilization of more The violet contribution of a blue colorant was defined as the area natural ingredients in food while keeping a vibrant color palette. under the visible absorption curve (AUC). The area was calculated Denish et al., Sci. Adv. 2021; 7 : eabe7871 7 April 2021 5 of 8 SCIENCE ADVANCES | RESEARCH ARTICLE using the left Riemann sum for integration, where the height is the set. For each species studied, 10 replicas were simulated using Hamiltonian absorbance and the width is the spectral resolution (for more spe- replica-exchange MD (HREMD) (52) as implemented in the PLUMED cific details, see method S1.1). plugin (53) (method S10.1). Ultraviolet-visible spectrometry Sequence alignment and construction of the phylogenetic Solutions were poured into relevant cuvette of 10-, 1-, and 0.1-mm tree of esterases of interest cell length, and then ultraviolet (UV)–visible absorption spectra (200 to Sequences tested in this study (table S11.1) were aligned using Geneious 800 nm) were recorded on a JASCO V-560 spectrophotometer. The 2017.10.1.3 using multiple sequence comparison by log-expectation solutions were kept at room temperature and protected from light (MUSCLE) alignment (method S11.1.). (method S6.1). The phylogenetic tree in Fig. 4C was constructed using the Geneious Consensus Tree Builder (method S11.1) and visualized using GraPhlAn Measurement of CD (https://github.com/siegel-lab-ucd/blue-pigment-publication.git) (54). CD was measured from 200 to 800 nm with a JASCO J-720 spec- trometer using the same solution for the measurement of stability Protein purification and activity screening of esterases described in section S6.1. To increase signal/noise ratio, the scan was An Escherichia coli codon-optimized gene encoding each protein was repeated four times, and the data were averaged (method S7.1) (21). purchased from Twist Biosciences and transferred into pET29b+ to encode a C-terminal hexahistidine tag. Mutant plasmids were pro- ESI-TOF-MS of aluminum complexes duced by Kunkel mutagenesis (55). Plasmids were incorporated into ESI-MS spectra were recorded with a Bruker COMPACT instrument E. coli BL21(DE3) via electroporation. Cultures were grown in Terrific and analyzed with the application purchased from Bruker Daltonics Broth at 37°C, induced with 1 mM isopropyl--d-thiogalactopyranoside, (method S7.1). and allowed to express at 18°C for 24 hours, after which cells were lysed, clarified, and protein-purified using gravity columns with immo- NMR measurement of aluminum complex of P2 bilized metal affinity chromatography, the details of which are provided NMR spectra were obtained with Bruker Daltonics AVANCE III HD in method S11.2. Proteins were screened for activity by combining 600 with a TCI cryoprobe and BBO cryoprobe (1H: 600 MHz and 10 l of RCE (100 mg/ml) with 90 l of 50 mM Hepes buffer (pH 13 C: 150 MHz) in a 5–mm–inside diameter tube at variable tempera- 7.5) and allowed to proceed at room temperature for 24 hours. Re- tures in D2O. Chemical shifts were recorded as parts per million (ppm) actions were quenched with 70% methanol and brought to pH 3 with using the proton resonance in the semi-heavy water (HDO) as a 1 l of HCl. Reactions were centrifuged at 4700 rpm for 3 min to standard (4.67 ppm). Various 1D and 2D measurements were car- remove insoluble protein. Analysis was done using the high-performance ried out (method S7.3). liquid chromatography (HPLC) method described in method S11.2. Geometric optimization of putative P2 and P5 3:1 complexes Gram-scale protein production with aluminum The M73H point mutant was created from the 1AUR (sequence ID Optimizations of either P2 or P5 molecules in a 3:1 complex with no. 46) wild-type (WT) plasmid via Kunkel mutagenesis (55) and then aluminum were performed, starting from relevant structures of mono- transformed into chemically competent E. coli BLR (DE3) cells (de- mers obtained from extensive MD simulations and analysis, as de- tails provided in method S11.3). The supernatant lysate containing tailed in section S10.1. These were arranged with a C3 symmetry active enzyme was collected and used for the reaction. The scaled-up around Al3+ and deprotonated at 3′ and 4′, and with Al3+ coordinated generation of P2 via the enzymatic reaction is described in method S11.4. to the oxygens at 3′ and 4′. The two chiral assemblies of the ligands, corresponding to left and right handed (labeled  and , respectively), P2 purification from enzyme-treated red cabbage were tried. Relaxations were then performed in two steps: (i) A pre- The material was purified using several techniques to remove impu- optimization was run in vacuum with CP2K (39), with DZVP-MOLOPT-­ rities: enzyme precipitation, solid-phase extraction, and preparatory SR-GTH basis set and GTH-BLYP pseudopotential (40–42) corrected HPLC (more detailed description is found in method S12.1). with the D3(0) Grimme dispersion (43). (ii) Final optimizations were run in Quantum Espresso (44, 45), using ultrasoft pseudopotentials Anthocyanin-metal complex [Al3+(P2−)3] formation details and the Perdew-Burke-Ernzerhof (PBE) exchange-correlation func- One-third equivalents of AlK(SO4)2 stock solution were added to the tional (46) with implicit solvent (Environ module) (47) in periodic aqueous P2 solution, adjusted to 7.0. The P2-Al complex solution cubic box of 80 bohr. The following parameters were adopted for the was transferred to a polypropylene container, immersed in liquid wavefunction convergence: kinetic-energy cutoff of 35 rydberg (Ry) nitrogen until fully frozen, and then placed into the lyophilizer with and charge density cutoff of 320 Ry. The Makov-Payne energy cor- vacuum set to 0.03 mbar and the condenser to −80°C. More detailed rection was used to remove finite-box effects (method S8.1). description is provided in method S12.2. 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